Publisher Correction: Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.

Perkinsus marinus in bioreactor: growth and a cost-reduced growth medium.

Perkinsus marinus (Perkinsea) is an osmotrophic facultative intracellular marine protozoan responsible for "Dermo" disease in the eastern oyster, Crassostrea virginica. In 1993 in vitro culture of P. marinus was developed in the absence of host cells. Compared to most intracellular protozoan parasites, the availability of P. marinus to grow in the absence of host cells has provided the basis to explore its use as a heterologous expression system. As the genetic toolbox is becoming available, there is also the need for larger-scale cultivation and lower-cost media formulations. Here, we took an industrial approach to scaled-up growth from a small culture flask to bioreactors, which required developing new cultivation parameters, including aeration, mixing, pH, temperature control, and media formulation. Our approach also enabled more real-time data collection on growth. The bioreactor cultivation method showed similar or accelerated growth rates of P. marinus compared to culture in T-flasks. Redox measurements indicated sufficient oxygen availability throughout the cultivation. Replacing fetal bovine serum with chicken serum showed no differences in the growth rate and a 60% reduction in the medium cost. This study opens the door to furthering P. marinus as a valid heterologous expression system by showing the ability to grow in bioreactors. ONE-SENTENCE SUMMARY: Perkinsus marinus, a microbial parasite of oysters that could be useful for developing vaccines for humans, has been shown to grow well in laboratory equipment that can be expanded to commercial scale using a less expensive growth formula than usual laboratory practice.

Genome to phenome tools: In vivo and in vitro transfection of Crassostrea virginica hemocytes.

The sequencing of the Crassostrea virginica genome has brought back the interest for gene delivery and editing methodologies. Here, we report the expression in oyster hemocytes of two heterologous expression vectors under the CMV promoter delivered with dendrimers. Expression was monitored using confocal microscopy, flow cytometry, and immunofluorescence assay. C. virginica hemocytes were able to express the green fluorescence protein and Crassostrea gigas vascular endothelial growth factor under CMV viral promoter both in vivo and in vitro. These results provide the bases for interrogating the genome and adapting genome editing methodologies.

Open Source Drug Discovery with the Malaria Box Compound Collection for Neglected Diseases and Beyond.

A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts.

The galectin CvGal1 from the eastern oyster (Crassostrea virginica) binds to blood group A oligosaccharides on the hemocyte surface.

The galectin CvGal1 from the eastern oyster (Crassostrea virginica), which possesses four tandemly arrayed carbohydrate recognition domains, was previously shown to display stronger binding to galactosamine and N-acetylgalactosamine relative to d-galactose. CvGal1 expressed by phagocytic cells is "hijacked" by the parasite Perkinsus marinus to enter the host, where it proliferates and causes systemic infection and death. In this study, a detailed glycan array analysis revealed that CvGal1 preferentially recognizes type 2 blood group A oligosaccharides. Homology modeling of the protein and its oligosaccharide ligands supported this preference over type 1 blood group A and B oligosaccharides. The CvGal ligand models were further validated by binding, inhibition, and competitive binding studies of CvGal1 and ABH-specific monoclonal antibodies with intact and deglycosylated glycoproteins, hemocyte extracts, and intact hemocytes and by surface plasmon resonance analysis. A parallel glycomic study carried out on oyster hemocytes (Kurz, S., Jin, C., Hykollari, A., Gregorich, D., Giomarelli, B., Vasta, G. R., Wilson, I. B. H., and Paschinger, K. (2013) J. Biol. Chem. 288) determined the structures of oligosaccharides recognized by CvGal1. Proteomic analysis of the hemocyte glycoproteins identified ß-integrin and dominin as CvGal1 "self"-ligands. Despite strong CvGal1 binding to P. marinus trophozoites, no binding of ABH blood group antibodies was observed. Thus, parasite glycans structurally distinct from the blood group A oligosaccharides on the hemocyte surface may function as potentially effective ligands for CvGal1. We hypothesize that carbohydrate-based mimicry resulting from the host/parasite co-evolution facilitates CvGal1-mediated cross-linking to ß-integrin, located on the hemocyte surface, leading to cell activation, phagocytosis, and host infection.

Survival and Detection of Bivalve Transmissible Neoplasia from the Soft-Shell Clam Mya arenaria (MarBTN) in Seawater.

Many pathogens can cause cancer, but cancer itself does not normally act as an infectious agent. However, transmissible cancers have been found in a few cases in nature: in Tasmanian devils, dogs, and several bivalve species. The transmissible cancers in dogs and devils are known to spread through direct physical contact, but the exact route of transmission of bivalve transmissible neoplasia (BTN) has not yet been confirmed. It has been hypothesized that cancer cells from bivalves could be released by diseased animals and spread through the water column to infect/engraft into other animals. To test the feasibility of this proposed mechanism of transmission, we tested the ability of BTN cells from the soft-shell clam (Mya arenaria BTN, or MarBTN) to survive in artificial seawater. We found that MarBTN cells are highly sensitive to salinity, with acute toxicity at salinity levels lower than those found in the native marine environment. BTN cells also survive longer at lower temperatures, with 50% of cells surviving greater than 12 days in seawater at 10 °C, and more than 19 days at 4 °C. With one clam donor, living cells were observed for more than eight weeks at 4 °C. We also used qPCR of environmental DNA (eDNA) to detect the presence of MarBTN-specific DNA in the environment. We observed release of MarBTN-specific DNA into the water of laboratory aquaria containing highly MarBTN-diseased clams, and we detected MarBTN-specific DNA in seawater samples collected from MarBTN-endemic areas in Maine, although the copy numbers detected in environmental samples were much lower than those found in aquaria. Overall, these data show that MarBTN cells can survive well in seawater, and they are released into the water by diseased animals. These findings support the hypothesis that BTN is spread from animal-to-animal by free cells through seawater.

CRISPR/Cas9 Ribonucleoprotein-Based Genome Editing Methodology in the Marine Protozoan Parasite Perkinsus marinus.

Perkinsus marinus (Perkinsozoa), a close relative of apicomplexans, is an osmotrophic facultative intracellular marine protozoan parasite responsible for "Dermo" disease in oysters and clams. Although there is no clinical evidence of this parasite infecting humans, HLA-DR40 transgenic mice studies strongly suggest the parasite as a natural adjuvant in oral vaccines. P. marinus is being developed as a heterologous gene expression platform for pathogens of medical and veterinary relevance and a novel platform for delivering vaccines. We previously reported the transient expression of two rodent malaria genes Plasmodium berghei HAP2 and MSP8. In this study, we optimized the original electroporation-based protocol to establish a stable heterologous expression method. Using 20 µg of pPmMOE[MOE1]:GFP and 25.0 × 106 P. marinus cells resulted in 98% GFP-positive cells. Furthermore, using the optimized protocol, we report for the first time the successful knock-in of GFP at the C-terminus of the PmMOE1 using ribonucleoprotein (RNP)-based CRISPR/Cas9 gene editing methodology. The GFP was expressed 18 h post-transfection, and expression was observed for 8 months post-transfection, making it a robust and stable knock-in system.

The Alveolate Perkinsus marinus: biological insights from EST gene discovery.

BACKGROUND: Perkinsus marinus, a protozoan parasite of the eastern oyster Crassostrea virginica, has devastated natural and farmed oyster populations along the Atlantic and Gulf coasts of the United States. It is classified as a member of the Perkinsozoa, a recently established phylum considered close to the ancestor of ciliates, dinoflagellates, and apicomplexans, and a key taxon for understanding unique adaptations (e.g. parasitism) within the Alveolata. Despite intense parasite pressure, no disease-resistant oysters have been identified and no effective therapies have been developed to date. RESULTS: To gain insight into the biological basis of the parasite's virulence and pathogenesis mechanisms, and to identify genes encoding potential targets for intervention, we generated>31,000 5' expressed sequence tags (ESTs) derived from four trophozoite libraries generated from two P. marinus strains. Trimming and clustering of the sequence tags yielded 7,863 unique sequences, some of which carry a spliced leader. Similarity searches revealed that 55% of these had hits in protein sequence databases, of which 1,729 had their best hit with proteins from the chromalveolates (E-value

A qPCR-Based Survey of Haplosporidium nelsoni and Perkinsus spp. in the Eastern Oyster, Crassostrea virginica in Maine, USA.

Eastern oyster (Crassostrea virginica) aquaculture is increasingly playing a significant role in the state of Maine's (USA) coastal economy. Here, we conducted a qPCR-based survey for Haplosporidium nelsoni, Perkinsus marinus, and Perkinsus chesapeaki in C. virginica (n = 1440) from six Maine sites during the summer-fall of 2016 and 2017. In the absence of reported die-offs, our results indicated the continued presence of the three protozoan parasites in the six sites. The highest H. nelsoni qPCR-prevalence corresponded to Jack's Point and Prentiss Island (x=40 and 48% respectively), both located in the Damariscotta River Estuary. Jack's Point, Prentiss Island, New Meadows River, and Weskeag River recorded the highest qPCR-prevalence for P. marinus (32-39%). While the P. marinus qPCR-prevalence differed slightly for the years 2016 and 2017, P. chesapeaki qPCR-prevalence in 2016 was markedly lower than 2017 (<20% at all sites versus >60% at all sites for each of the years, respectively). Mean qPCR-prevalence values for P. chesapeaki over the two-year study were ≥40% for samples from Jack's Point (49%), Prentiss Island (44%), and New Meadows River (40%). This study highlights that large and sustained surveys for parasitic diseases are fundamental for decision making toward the management of the shellfish aquaculture industry, especially for having a baseline in the case that die-offs occur.

Molecular Epizootiology of Toxoplasma gondii and Cryptosporidium parvum in the Eastern Oyster (Crassostrea virginica) from Maine (USA).

Shellfish are known as a potential source of Toxoplasma gondii (responsible for toxoplasmosis), and Cryptosporidium parvum, which is one of the major causes of gastroenteritis in the world. Here we performed a comprehensive qPCR-based monthly survey for T. gondii and C. parvum during 2016 and 2017 in oysters (Crassostrea virginica) (n = 1440) from all six sites along the coast of Maine (USA). Pooled samples (mantle, gills, and rectum) from individual oysters were used for DNA extraction and qPCR. Our study resulted in detections of qPCR positives oysters for T. gondii and C. parvum at each of the six sites sampled (in 31% and 10% of total oysters, respectively). The prevalence of T. gondii was low in 2016, and in September 2017 several sites peaked in prevalence with 100% of the samples testing positive. The prevalence of C. parvum was very low except in one estuarine location (Jack's Point) in June 2016 (58%), and in October of 2016, when both prevalence and density of C. parvum at most of the sampling sites were among the highest values detected. Statistical analysis of environmental data did not identify clear drivers of retention, but there were some notable statistically significant patterns including current direction and nitrate along with the T. gondii prevalence. The major C. parvum retention event (in October 2016) corresponded with the month of highest dissolved oxygen measurements as well as a shift in the current direction revealed by nearby instrumentation. This study may guide future research to locate any contributing parasite reservoirs and evaluate the potential risk to human consumption.

From the raw bar to the bench: Bivalves as models for human health.

Bivalves, from raw oysters to steamed clams, are popular choices among seafood lovers and once limited to the coastal areas. The rapid growth of the aquaculture industry and improvement in the preservation and transport of seafood have enabled them to be readily available anywhere in the world. Over the years, oysters, mussels, scallops, and clams have been the focus of research for improving the production, managing resources, and investigating basic biological and ecological questions. During this decade, an impressive amount of information using high-throughput genomic, transcriptomic and proteomic technologies has been produced in various classes of the Mollusca group, and it is anticipated that basic and applied research will significantly benefit from this resource. One aspect that is also taking momentum is the use of bivalves as a model system for human health. In this review, we highlight some of the aspects of the biology of bivalves that have direct implications in human health including the shell formation, stem cells and cell differentiation, the ability to fight opportunistic and specific pathogens in the absence of adaptive immunity, as source of alternative drugs, mucosal immunity and, microbiome turnover, toxicology, and cancer research. There is still a long way to go; however, the next time you order a dozen oysters at your favorite raw bar, think about a tasty model organism that will not only please your palate but also help unlock multiple aspects of molluscan biology and improve human health.

Perkinsus marinus superoxide dismutase 2 (PmSOD2) localizes to single-membrane subcellular compartments.

Perkinsus marinus (Phylum Perkinsozoa), a protozoan parasite of oysters, is considered one of the earliest diverging groups of the lineage leading to dinoflagellates. Perkinsus trophozoites are phagocytosed by oyster hemocytes, where they are likely exposed to reactive oxygen species. As part of its reactive oxygen detoxifying pathway, P. marinus possesses two iron-cofactored SOD (PmSOD1 and PmSOD2). Immunoflourescence analysis of P. marinus trophozoites and gene complementation in yeast revealed that PmSOD1 is targeted to the mitochondria. Surprisingly, although PmSOD2 is characterized by a bipartite N-terminus extension typical of plastid targeting, in preliminary immunofluorescence studies it was visualized as punctuate regions in the cytoplasm that could not be assigned to any organelle. Here, we used immunogold electron microscopy to examine the subcellular localization PmSOD2 in P. marinus trophozoites. Gold grains were mostly associated with single-membrane vesicle-like structures, and eventually, localized to electron-dense, apparently amorphous material present in the lumen of a larger, unique compartment. The images suggested that PmSOD2 is targeted to small vesicles that fuse and/or discharge their content into a larger compartment, possibly the large vacuole typical of the mature trophozoites. In light of the in silico targeting prediction, the association of PmSOD2 with single-membrane compartments raises interesting questions regarding its organellar targeting, and the nature of a putative relic plastid in Perkinsus species.

Transfection of the protozoan parasite Perkinsus marinus.

Ongoing efforts for sequencing the genome of the protozoan parasite Perkinsus marinus, together with functional genomic initiatives, have continued to provide invaluable information about genes and metabolic pathways that not only will increase our understanding of its biology, but also have the potential to reveal useful targets for intervention. The lack of molecular tools for the functional characterization of genes of interest, however, has hindered progress in this regard. Here we report the development and validation of transfection methodology for this parasite. We first selected from our P. marinus EST collection a highly expressed gene, which we designated "MOE" (PmMOE), to which we fused at the C-terminus the enhanced green fluorescent protein (GFP) as a reporter gene (pPmMOE-GFP). The exogenous DNA was introduced into the trophozoite stage of the parasite by electroporation using the Nucleofector technology. The transfection efficiency was 37.8% with fluorescence detected as early as 14 h after electroporation, with the transfectants still remaining fluorescent after 8 months even in the absence of drug selection. The 5' flanking region was essential for transcription; constructs with 100 and 204 bp flanking the transcription start site also drove transcription effectively. Polymerase chain reaction (PCR) and Southern blot analyses was consistent with integration by non-homologous recombination. This transfection technique, the first one reported for a member of the Perkinsozoa, provides a new tool for studies of gene regulation and expression, protein targeting, and protein-protein interactions, and should significantly contribute to gain further insight into the biology of Perkinsus spp.

Assessment of the northern distribution range of selected Perkinsus species in eastern oysters (Crassostrea virginica) and hard clams (Mercenaria mercenaria) with the use of PCR-based detection assays.

Perkinsus species are protistan parasites of molluscs. In Chesapeake Bay, Perkinsus marinus, Perkinsus chesapeaki, and Perkinsus andrewsi are sympatric, infecting oysters and clams. Although P. marinus is a pathogen for Crassostrea virginica, it remains unknown whether P. andrewsi and P. chesapeaki are equally pathogenic. Perkinsus species have been reported in C. virginica as far north as Maine, sometimes associated with high prevalence, but low mortality. Thus, we hypothesized that, in addition to P. marinus, Perkinsus species with little or no pathogenicity for C. virginica may be present. Accordingly, we investigated the distribution of Perkinsus species in C. virginica and Mercenaria mercenaria, collected from Maine to Virginia, by applying PCR-based assays specific for P. marinus, P. andrewsi, and a Perkinsus sp. isolated from M. mercenaria. DNA samples of M. mercenaria possessed potent PCR inhibitory activity, which was overcome by the addition of 1 mg/ml BSA and 5% (v/v) DMSO to the PCR reaction mixture. All 3 Perkinsus species were found in both host species throughout the study area. Interestingly, the prevalence of P. marinus in M. mercenaria was significantly lower than in C. virginica, suggesting that M. mercenaria is not an optimal host for P. marinus.

An Agar-Based Method for Plating Marine Protozoan Parasites of the Genus Perkinsus.

The genus Perkinsus includes protozoan parasites of mollusks responsible for losses in the aquaculture industry and hampering the recovery of natural shellfish beds worldwide, and they are a key taxon for understanding intracellular parasitism adaptations. The ability to propagate the parasite in liquid media, in the absence of the host, has been crucial for improving understanding of its biology; however, alternative techniques to grow the parasite are needed to explore other basic aspects of the Perkinsus spp. biology. We optimized a DME: Ham's F12-5% FBS- containing solid agar medium for plating Perkinsus marinus. This solid medium supported trophozoite propagation both by binary fission and schizogony. Colonies were visible to the naked eye 17 days after plating. We tested the suitability of this method for several applications, including the following: 1) Subcloning P. marinus isolates: single discrete P. marinus colonies were obtained from DME: Ham's F12-5% FBS- 0.75% agar plates, which could be further propagated in liquid medium; 2) Subcloning engineered Perkinsus mediterraneus MOE[MOE]: GFP by streaking cultures on plates; 3) Chemical susceptibility: Infusing the DME: Ham's F12-5% FBS- 0.75% agar plates with triclosan resulted in inhibition of the parasite propagation in a dose-dependent manner. Altogether, our plating method has the potential for becoming a key tool for investigating diverse aspects of Perkinsus spp. biology, developing new molecular tools, and for biotechnological applications.

Identification of MMV Malaria Box inhibitors of Perkinsus marinus using an ATP-based bioluminescence assay.

"Dermo" disease caused by the protozoan parasite Perkinsus marinus (Perkinsozoa) is one of the main obstacles to the restoration of oyster populations in the USA. Perkinsus spp. are also a concern worldwide because there are limited approaches to intervention against the disease. Based on the phylogenetic affinity between the Perkinsozoa and Apicomplexa, we exposed Perkinsus trophozoites to the Medicines for Malaria Venture Malaria Box, an open access compound library comprised of 200 drug-like and 200 probe-like compounds that are highly active against the erythrocyte stage of Plasmodium falciparum. Using a final concentration of 20 µM, we found that 4 days after exposure 46% of the compounds were active against P. marinus trophozoites. Six compounds with IC50 in the µM range were used to compare the degree of susceptibility in vitro of eight P. marinus strains from the USA and five Perkinsus species from around the world. The three compounds, MMV666021, MMV665807 and MMV666102, displayed a uniform effect across Perkinsus strains and species. Both Perkinsus marinus isolates and Perkinsus spp. presented different patterns of response to the panel of compounds tested, supporting the concept of strain/species variability. Here, we expanded the range of compounds available for inhibiting Perkinsus proliferation in vitro and characterized Perkinsus phenotypes based on their resistance to six compounds. We also discuss the implications of these findings in the context of oyster management. The Perkinsus system offers the potential for investigating the mechanism of action of the compounds of interest.

Protozoan parasites of bivalve molluscs: literature follows culture.

Bivalve molluscs are key components of the estuarine environments as contributors to the trophic chain, and as filter -feeders, for maintaining ecosystem integrity. Further, clams, oysters, and scallops are commercially exploited around the world both as traditional local shellfisheries, and as intensive or semi-intensive farming systems. During the past decades, populations of those species deemed of environmental or commercial interest have been subject to close monitoring given the realization that these can suffer significant decline, sometimes irreversible, due to overharvesting, environmental pollution, or disease. Protozoans of the genera Perkinsus, Haplosporidium, Marteilia, and Bonamia are currently recognized as major threats for natural and farmed bivalve populations. Since their identification, however, the variable publication rates of research studies addressing these parasitic diseases do not always appear to reflect their highly significant environmental and economic impact. Here we analyzed the peer- reviewed literature since the initial description of these parasites with the goal of identifying potential milestone discoveries or achievements that may have driven the intensity of the research in subsequent years, and significantly increased publication rates. Our analysis revealed that after initial description of the parasite as the etiological agent of a given disease, there is a time lag before a maximal number of yearly publications are reached. This has already taken place for most of them and has been followed by a decrease in publication rates over the last decade (20- to 30- year lifetime in the literature). Autocorrelation analyses, however, suggested that advances in parasite purification and culture methodologies positively drive publication rates, most likely because they usually lead to novel molecular tools and resources, promoting mechanistic studies. Understanding these trends should help researchers in prioritizing research efforts for these and other protozoan parasites, together with their development as model systems for further basic and translational research in parasitic diseases.

Quantitative assessment of the proliferation of the protozoan parasite Perkinsus marinus using a bioluminescence assay for ATP content.

Perkinsus marinus is a protozoan parasite that causes "Dermo" disease in the eastern oyster Crasssostrea virginica in coastal areas of the USA. Until now, intervention strategies against the parasite have found limited success, and Dermo still remains one of the main hurdles for the restoration of oyster populations. We adapted a commercial adenosine tri-phosphate (ATP) content-based assay to assess the in vitro proliferation of P. marinus in a 96-well plate format, and validated the method by measuring the effects of potential anti-proliferative compounds. The sensitivity (1.5-3.1 × 10(4) cells/well), linearity (R (2) = 0.983), and signal stability (60 min) support the reliability of the assay for assessing cell proliferation. Validation of the assay by culturing P. marinus in the presence of increasing concentrations of triclosan showed a dose-response profile. The IC50 value obtained was higher than that reported earlier, possibly due to the use of different viability assay methods and a different P. marinus strain. The antibiotics G418 and tetracycline and the herbicide fluridone were active against P. marinus proliferation; the IC50 of chloramphenicol, ciprofloxacin, and atrazine was relatively high suggesting either off-target effects or inability to reach the targets. The validation of the ATP-based assay, together with significant advantages of the Perkinsus culture methodology (homogeneity, reproducibility, and high cell densities), underscores the value of this assay for developing high-throughput screens for the identification of novel leader compounds against Perkinsus species, and most importantly, for the closely-related apicomplexan parasites.